Martin S. Davey

Human neutrophil clearance of bacterial pathogens triggers anti-microbial γδ T cell responses in early infection

Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early infection and suggest novel diagnostic and therapeutic approaches.

Failure to detect production of IL-10 by activated human neutrophils.

To the Editor: We read with great interest the paper by De Santo et al.1 published in the November 2010 issue of Nature Immunology, which has received wide attention because of its potential implications for inflammation and immunotherapy. The authors show that the acute-phase protein serum amyloid A1 (SAA-1) induces substantial secretion of the immunosuppressive cytokine interleukin 10 (IL-10) by human neutrophils (up to 100–400 ng/ml) and that invariant natural killer T cells (iNKT cells) are able to diminish this. The authors conclude that harnessing iNKT cells might “be useful therapeutically by decreasing the frequency of immunosuppressive neutrophils and restoring tumor-specific immune responses” in patients with melanoma and other patients with high plasma SAA-1 concentrations1. Intimate crosstalk of neutrophils and monocytes with another population of ‘unconventional’ human T cells, Vg9Vd2 gd T cells, has been demonstrated2. Of relevance for immunotherapy, Vg9Vd2 T cells are much more abundant in human blood than are iNKT cells and can readily be activated by aminobisphosphonate drugs such as zoledronate (Zometa). Small-scale trials have shown promising results for therapy targeting Vg9Vd2 T cells to treat metastatic disease in patients with multiple myeloma, prostate cancer or breast cancer3. Thus, we were keen to ‘translate’ the described interaction of iNKT cells with neutrophils to our own experimental system2,4 and to test whether activated Vg9Vd2 T cells likewise inhibits IL-10-producing neutrophils. Unexpectedly, we found that highly purified, obtained by negative selection human neutrophils (Supplementary Fig. 1) did not secrete IL-10 (Fig. 1a) or express IL-10 mRNA (Fig. 1b) in response to recombinant SAA-1 at the same concentrations used by De Santo et al.1 Notably, and in contrast to De Santo et al., we also did not detect IL-10 in the culture supernatants of neutrophils stimulated with either Salmonella abortus equi lipopolysaccharide (LPS) or ultrapure Escherichia coli LPS (Fig. 1 and Supplementary Fig. 2). The failure to detect IL-10 production by human neutrophils in response to SAA-1 or LPS was reproducible in four independent laboratories at Cardiff (UK), Verona (Italy), Milan (Italy) and Essen (Germany) and was independent of the isolation procedure, the method used to lyse red blood cells, the culture dishes used, cell numbers and medium supplements (Supplementary Methods). The lack of IL-10 in neutrophil culture supernatants was consistent with the absence of both IL-10 mRNA (Fig. 1b) and IL-10 protein (data not shown) in total lysates of LPS-stimulated neutrophils, which confirmed published data5,6. Finally, human neutrophils did not secrete IL-10 even when incubated with other Toll-like receptor agonists (poly(I:C), Pam3CSK4 or R-848), cytokines (IFN-g, TNF, GM-CSF or G-CSF), curdlan, neutrophil-activating protein derived from Helicobacter pylori, the chemoattractant fMLF or insoluble immunocomplexes (Supplementary Fig. 2 and data not shown). In their study, De Santo et al. positively select cells on the basis of CD11b expression and define them as >95% CD11b+CD15+ (ref. 1), compared with the value of over 98–99% achieved in our laboratories2,4,7,8 (Supplementary Methods). Of note, both peripheral neutrophils and monocytes expressed CD11b. Although neutrophils were distinctly CD15+, monocytes had intermediate surface expression of CD15 (Supplementary Fig. 1). Contaminating monocytes may thus give rise to false-positive data in whole-population analyses by enzyme-linked immunosorbent assay (ELISA), quantitative RT-PCR or immunoblot analysis. When we added

Activation of Human γδ T Cells by Cytosolic Interactions of BTN3A1 with Soluble Phosphoantigens and the Cytoskeletal Adaptor Periplakin

The three butyrophilin BTN3A molecules, BTN3A1, BTN3A2, and BTN3A3, are members of the B7/butyrophilin-like group of Ig superfamily receptors, which modulate the function of T cells. BTN3A1 controls activation of human Vγ9/Vδ2 T cells by direct or indirect presentation of self and nonself phosphoantigens (pAg). We show that the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate binds to the intracellular B30.2 domain of BTN3A1 with an affinity of 1.1 μM, whereas the endogenous pAg isopentenyl pyrophosphate binds with an affinity of 627 μM. Coculture experiments using knockdown cell lines showed that in addition to BTN3A1, BTN3A2 and BTN3A3 transmit activation signals to human γδ T cells in response to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and the aminobisphosphonate drug zoledronate that causes intracellular accumulation of isopentenyl pyrophosphate. The plakin family member periplakin, identified in yeast two-hybrid assays, interacted with a membrane-proximal di-leucine motif, located proximal to the B30.2 domain in the BTN3A1 cytoplasmic tail. Periplakin did not interact with BTN3A2 or BTN3A3, which do not contain the di-leucine motif. Re-expression into a BTN3A1 knockdown line of wild-type BTN3A1, but not of a variant lacking the periplakin binding motif, BTN3A1Δexon5, restored γδ T cell responses, demonstrating a functional role for periplakin interaction. These data, together with the widespread expression in epithelial cells, tumor tissues, and macrophages detected using BTN3A antiserum, are consistent with complex functions for BTN3A molecules in tissue immune surveillance and infection, linking the cell cytoskeleton to γδ T cell activation by indirectly presenting pAg to the Vγ9/Vδ2 TCR.

Microbe-Specific Unconventional T Cells Induce Human Neutrophil Differentiation into Antigen Cross-Presenting Cells

The early immune response to microbes is dominated by the recruitment of neutrophils whose primary function is to clear invading pathogens. However, there is emerging evidence that neutrophils play additional effector and regulatory roles. The present study demonstrates that human neutrophils assume Ag cross-presenting functions and suggests a plausible scenario for the local generation of APC-like neutrophils through the mobilization of unconventional T cells in response to microbial metabolites. Vγ9/Vδ2 T cells and mucosal-associated invariant T cells are abundant in blood, inflamed tissues, and mucosal barriers. In this study, both human cell types responded rapidly to neutrophils after phagocytosis of Gram-positive and Gram-negative bacteria producing the corresponding ligands, and in turn mediated the differentiation of neutrophils into APCs for both CD4+ and CD8+ T cells through secretion of GM-CSF, IFN-γ, and TNF-α. In patients with acute sepsis, circulating neutrophils displayed a similar APC-like phenotype and readily processed soluble proteins for cross-presentation of antigenic peptides to CD8+ T cells, at a time when peripheral Vγ9/Vδ2 T cells were highly activated. Our findings indicate that unconventional T cells represent key controllers of neutrophil-driven innate and adaptive responses to a broad range of pathogens.

Neutrophils are important in early control of lung infection by Yersinia pestis

In this paper we evaluate the role of neutrophils in pneumonic plague. Splenic neutrophils from naïve BALB/c mice were found to reduce numbers of culturable Yersinia pestis strain GB in suspension. A murine, BALB/c, intranasal model of pneumonic plague was used in conjunction with in vivo neutrophil ablation, using the GR-1 antibody. This treatment reduced neutrophil numbers without affecting other leukocyte numbers. Neutrophil ablated mice exhibited increased bacterial colonisation of the lung 24h post infection. Furthermore, exposure of Y. pestis to human neutrophils resulted in a 5-fold reduction in the number of viable bacterial cells, whereas, PBMCs had no effect.

Human γδ T-cell responses in infection and immunotherapy: Common mechanisms, common mediators?

Upon receiving the Nobel Prize in Physiology or Medicine in 1987, Susumu Tonegawa referred to the then recent discovery of the γδ T‐cell receptor and stated that “while the function of the T cells bearing this receptor is currently unknown (…) these T cells may be involved in an entirely new aspect of immunity”. [Tonegawa, S., Scand. J. Immunol. 1993. 38: 303–319]. Twenty‐five years of intense research later this ambivalent view still holds true. Immunologists now appreciate that γδ T cells indeed represent a highly intriguing “new aspect of immunity” that is unique and distinct from conventional lymphocytes, yet even scientists in the field still struggle to understand the molecular basis of γδ T‐cell responses, especially with respect to the enigmatic mode of antigen recognition. Here, we portray the peculiar responsiveness of human Vγ9/Vδ2 T cells to microorganisms, tumor cells and aminobisphosphonates, in an attempt to integrate the corresponding — and at times confusing — findings into a “theory of everything” that may help explain how such diverse stimuli result in similar γδ T‐cell responses via the recognition of soluble low molecular weight phosphoantigens.

Human gamma delta T-cell responses in infection and immunotherapy: Common mechanisms, common mediators?

Upon receiving the Nobel Prize in Physiology or Medicine in 1987, Susumu Tonegawa referred to the then recent discovery of the γδ T‐cell receptor and stated that “while the function of the T cells bearing this receptor is currently unknown (…) these T cells may be involved in an entirely new aspect of immunity”. [Tonegawa, S., Scand. J. Immunol. 1993. 38: 303–319]. Twenty‐five years of intense research later this ambivalent view still holds true. Immunologists now appreciate that γδ T cells indeed represent a highly intriguing “new aspect of immunity” that is unique and distinct from conventional lymphocytes, yet even scientists in the field still struggle to understand the molecular basis of γδ T‐cell responses, especially with respect to the enigmatic mode of antigen recognition. Here, we portray the peculiar responsiveness of human Vγ9/Vδ2 T cells to microorganisms, tumor cells and aminobisphosphonates, in an attempt to integrate the corresponding — and at times confusing — findings into a “theory of everything” that may help explain how such diverse stimuli result in similar γδ T‐cell responses via the recognition of soluble low molecular weight phosphoantigens.

A Promising Target for Treatment of Multidrug-Resistant Bacterial Infections

Multidrug-resistant infections pose a major quandary for clinicians by complicating therapy choice, compromising patient recovery, and creating a serious threat to public health ([1][1]). In Gram-negative bacteria, resistance to almost all β-lactam antibiotics, including the carbapenems, can be

HmgR, a key enzyme in the mevalonate pathway for isoprenoid biosynthesis, is essential for growth of Listeria monocytogenes EGDe

Isoprenoids may be synthesized via one of two pathways, the classical mevalonate pathway or the alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. While the majority of bacteria utilize a single pathway for isoprenoid biosynthesis, Listeria monocytogenes is unusual in possessing the complete set of genes for both pathways. Here, we utilized new molecular tools to create precise gene deletions in selected genes encoding enzymes of both pathways, gcpE, lytB (encoding proteins in the MEP pathway) and hmgR (encoding a protein in the mevalonate pathway). We demonstrate that the hmgR gene can only be deleted when the growth medium is supplemented with exogenous mevalonate. Furthermore, full growth of the mutant in the absence of mevalonate was only possible when the intact hmgR gene was supplied in trans using an IPTG-inducible expression system. Murine competitive index assays performed via the oral and intraperitoneal routes of infection revealed that the mevalonate hmgR mutant could not be recovered from livers and spleens 3 days post-infection. We propose that HmgR in L. monocytogenes EGDe is involved in essential metabolic functions and that an intact MEP pathway is not capable of sustaining growth.

Yersinia pseudotuberculosis is resistant to killing by human neutrophils

The interaction between human neutrophils and the Gram negative gastrointestinal pathogen Yersinia pseudotuberculosis was investigated in vitro. Despite the wealth of data describing how Yersinia can affect the function of neutrophils, there are no published studies describing if neutrophil cells can affect the viability of Y. pseudotuberculosis. The wild-type IP32953 strain of Y. pseudotuberculosis was found to be resistant to killing by human neutrophils. Confocal examination and flow-cytometric analysis of this interaction revealed that bacteria were taken up.